The combination of genome sequencing along with commonality of genetic function across species is providing an opportunity to characterize the genes of higher organisms in two ways: by analyzing site-specific changes in homologous genes in model systems and through analysis of higher eukaryotic genes cloned within model organisms. The yeast Saccharomyces cerevisiae has proven ideal for many cross-species studies. The growing body of knowledge and techniques that have made it the best-characterized eukaryotic genome (Dujon, Trends Genet. 12, 263-270, 1996; Hieter et al., Nat. Genet 13, 253-255, 1996; Resnick and Cox, Mutat. Res. 451, 1-11, 2000; Shashikant et al., Gene 223, 9-20, 1998) also allow the experimental manipulation of large heterologous genomic DNA fragments cloned into yeast artificial chromosomes (YACs) (Anand, Trends Biotechnol. 10, 35-40, 1992, Larionov et al, Proc. Natl. Acad. Sci. USA 94, 7384-7387, 1997; Brown et al., Trends Biotechnol. 18, 218-23, 2000).
While it is possible in yeast to modify natural chromosomes or YACs without leaving behind any heterologous sequence, present systems have limited flexibility and are laborious. For example, null mutations are usually made in yeast by gene replacement, such that a marker gene replaces the sequence that is deleted (Wach et al., Yeast 10, 1793-1808, 1994). Marker recycling procedures, based on homologous or site-specific recombination or religation of DNA ends, also leave heterologous material at the deleted locus, such as hisG (Alani et al, Genetics 116, 541-545, 1987), FRT (Storici et al., Yeast 15, 271-283, 1999), loxP (Delneri et al., Gene 252, 127-135, 2000), or I-SceI (Fairhead et al., Yeast 12, 1439-1457, 1996) sequences. To accomplish sequence modification such that no heterologous material is retained requires subcloning and in vitro mutagenesis (Scherer and Davis, Proc. Natl. Acad. Sci. USA 76, 4949-4955, 1979; Barton et al., Nucleic Acids Res. 18, 7349-7355, 1990). PCR-based procedures that do not involve cloning are inefficient or require multistep reactions which increase the risk of additional mutations (Langle-Rouault and Jacobs, Nucleic Acids Res. 23, 3079-3081, 1995; Erdeniz et al., Genome Res. 7, 1174-1183, 1997). An alternative approach has been demonstrated in yeast for the CYC1 gene that relies on transformation with an oligonucleotide (Moerschell et al., Proc. Natl. Acad. Sci. USA 85, 524-528, 1988), but the method is restricted to the generation of mutants with a selectable phenotype and appears to be target dependent (Kmiec, “Targeted gene repair” [editorial], Gene Ther. 6, 1-3, 1999). Oligonucleotides, when combined with gap repair, can also be used to modify plasmids in yeast (Duno et al., Nucleic Acids Res. 27:e1, 1999); however, this approach is limited by restriction site availability.